In vivo dose response assays

Tumor assays

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1. Tumor grow th measurem ents; tumor growth delay.

 After irradiation, the tum o r is meas ured daily to determ in e the mean diam eter, or volum e.

 Plot tumor size v e rsus time after treat ment.

 Unirradiated tu m o rs will grow continuously.

 Irradiated tum o rs will show some shri nkage or delayed growth, then regrow.

 Score growth delay or time to grow to a specified size ; plot versus dose.

 Dose Modification Factor: use of growth or growth delay curves to meas ure the effect of an added agent or treat ment e.g., a radiosensitizer .

2. Tumor Control (TCD 50 ass a y)

 Irradiate tum o rs of uniform sizes with various doses. Observe for local control or recurrence. Plot % control vs. dose.

 TCD 50 = dose to control 50% of tum o rs

 This is a m o re relev a nt assay for radiothe rapy than growth del a y, but requires keeping greater num b ers of anim als for longer periods of tim e so is more costly.

1, 2 or 10 doses: 24 hours betw een fractions, shift in TCD 50 i ndicates an extensive repair of sublethal damage.

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3. Dilution assay te chnique

 Inject a known num ber of leukem i a cells from a donor m ouse into a recipient m ouse to determ ine the num ber of cells needed to cause leukemi a.

 TD 50 = nu mb er of cells needed to cau se leukem i a in 50% of recipient ani m als.

 Irradiate donor m ouse, collect and count l e ukem i c cells, inject i.p. into recipient m ouse, determ ine TD 50 .

 Surviving fraction = (Control TD 50 )(Irradiated TD 50 )

Dose response in vivo : dilution assay technique for various m u rine tum o rs plus/m inus oxygen.

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Dilution assay:

 Various num bers of tum o r cells from donor anim al injected into groups of recipient animals.

 A determ ination of t h e num ber of cells required to i nduce tum o rs in 50% of the recipients is made (TD 50 ).

 Control TD 50 /test TD 50 = SF

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4. Lung colony assay

 Sam e idea as the dilution assay, except uses solid tum o r cells rather than leukem i a cells.

 The tu mor is irradiated in a do nor anim al, rem oved and single cell suspension prepared, cells inject ed into recipient an imal.

 About 20 days later, lung colonies are counted.

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Lung colony assay system:

 Tum o r irradiated in situ , then excised and mad e into single cell suspension.

 Known num bers of cells injected i.v.

 The num b er of lung colonies is a measure of the num ber of clonogenic tum o r cells in the injected suspension.

 Co mpare to control; plot survival curve

 Mist inject same total num ber o f cells: m i x cells with large num b e r of “carrier” h eavily irradiated cells.

5. In Vivo-In Vitro ass ay

 Som e tu mor cell lines have been adapted to grow both in vivo and in vitro .

 Irradiate tum o rs in animals, rem ove tumors, prepare single-cell suspension, plate cells in suitable m e dium for colony form ation.

 There is n o t necessarily qualita tive or quantitative agreement between results of this assay and results obtained when tum o rs are left in situ . Possible reasons include PLDR or differences in types of da mages expressed.

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Xenografts - tum o r growing in a different species host. Must suppress host imm u me system :

 nude m i ce: lack thym us

 scid mice: lack B and T cells

 radiation or drugs can suppress imm une system

Advantages

 Cells retain the human karyotype a nd some dose-response characteristics of the tumor from which they were derived.

 For many tum o rs growth delay and c linical rem i ssion rate correlate.

Disadvantages

 Possible rejection by host, may cause mi sleading results, especially if the endpoint i s tum o r control.

 Cells may change; kinetic changes, selection.

 Host is different, absence of an imm u ne response is artificial.

 Strom a l tissue different, rodent origi n , makes studies where vascularity is im portant questionable.

6 . Spheroids

An in vitro tum o r model system

Certain cell types growing in suspension or in soft agar will agg r egate and rem a in in contact, form ing m u lticellular, 3-D sp heres called spheroids.

Spheroi d s rely on diffusion of O 2 and nutrients from the media. As they grow larger, they will develop nutrie n t-deprived centers.

Useful as a m odel of m i cro m etastatic tu mors

Larger spheroids generally cont ain 3 types of cell populations

 Asynchronous, cycling cells

 Noncycling cells

 Noncycling, hypoxic cells

Growth methods

 Spinner flasks

 Liquid ove rlay

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Advantages

 Diffusion (O 2 , s m all mol ecules, radiolabelled agents, M A b)

 nutrient de prived centers

 hypoxia in center

 m odel for tum o rs

 can be irradiated

 growth can be m onitored

 disaggregate for anal ysis of cell survival

 cells in spheroid m o re radioresistant than same cells in vitro , small spheroids may have the same D 0 as single cells.

Normal Tissues: Dose res p onse as says

Three b a sic types

 direct clonogenic assay

 functional assay

 m u ltifraction experiments used to a ssemble dose-response relationshi p or

α / β ratios

Direct cl onogeni c ass a ys

S k in Clones Assay

 Thirty-plus years ag o, skin damage wa s a major concern in radiation the r apy because of the use of orthovoltage X -ray machine. Now, skin damage is mu ch less of a clinical issue due to the us e of linacs and MeV energy phot ons.

 Technique: Pluck hair from m o use’s back , cover center of plucked area with a lead disk, irradiated surrounding area with high dose (e.g. 30 Gy) to create a mo at of dead cells.

 Remov e lead disk and irradiate centr al test area with varying doses.

 After tim e has been allowed for the skin to regrow, count patches, or islands, of skin regrowth.

 Each patch represents a clone f o rmed from a single surviving stem cell.

 Construct a survival curve by plotting nu m b er of surviving clones vs. dose.

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N.B. Actu al nu mber of cells in skin region is not known, only the num ber of colonies. Width of shoulder can be estimated with split dose experim e nt. Separation of the two lines is D q .

Crypt cell ass a y

 Crypt cells constantly dividing

 These are the renewing stem cells in the intestinal epithelium

Procedure

 Deliver a total body dose of 11-16 Gy, which kills m o st crypt cells but spares the cells in the villi.

 As villi cells are lost by norm a l migration and sloughing off processes, no functional cells replace them : the villi shrink.

 At 3-4 days post irra diation the crypts begin to regenerate.

 Animals s acrificed, cross

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sections of intestines stained and scored.

 Plot number of regenerating cr ypts per circumference vs dose.

Sim ilar assays can be used for test es stem cells and kidney tubules.

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Crypt cell survival curves, si ngle dose an d m u ltiple doses

 D 0 for 1 fraction = 1.3 Gy.

 Shoul der is large: D q = 4-4.5 Gy (large capacity for repair).

Lim itations:

 Does not directly m e asure survi v ing fracti on.

 Useful dose range 11-16 Gy: need enough kill to measure individual regenerating crypts.

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Cells transplanted to another site

Spleen col o ny assay

 Sim ilar to dilution assay except uses normal bone marrow cells, rather than leukemia, and colonies form in the spleen.

 To conduct experim e nts, irradiate donor mouse to different doses, rem o ve bone marrow cells, count, and inject known num ber of cells into supra- lethally irradiated m i ce (spleens sterilized).

 9-10 days later rem o ve sp leen and count nodules.

 Calculate survi v ing fraction as SF = (# colonies counted)/(# cells inoculated x “PE”). Plot SF ve rsus dose for survival curve.

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Recipient mi ce tr eat ed with 9 Gy, supralethal dose sterilizes spleen cells.

~ 100 cells injected to form 1 spleen colony.

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Note that the norm a l bone marrow cells are relatively radiation sensitive, with both a small D 0 and sm all n.

Spleen colony assay for bone marrow cells

Fat pad assay

Normal thyroid gland cells Also used for m a mmary gland

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24 hour delay before re m oving t hyroi d gland reveals rep a ir of potentially lethal damage (PLD).

Summary of n o rmal tissu e s e nsiti v ity

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Hu man A-T cells ar e a DNA repair-deficie nt m u tant: one of the m o st radiation- sensitive human cell lines.

BM-CF U, mammali an thyroid cells: transplant into another tissue in recip i ent ani m al for assay.

Shoul der width is the principal variable .

Normal Tissues: Functional assays

Not a direct measure of cell survival, but direct relevance to clinical side effects.

Sk in reaction Pig S k in

Very sim i lar to human skin:

Color, hair follicles, sweat glands subcutaneous fat.

 Set up an arbitrary scal e for scoring skin reactions.

 Irradiate skin regions with various doses.

 Score skin reaction as a function of time.

Two waves of skin reaction Early: 10-40 days

Late: gradual increase to a broad maxim u m 50-100 days , m o re severe dam a ge.

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Dose resp onse is obtained by plotting skin reaction vs dose. Fractionation results in considerable sparing.

Other Functional Endpoints

A variety of other functiona l assays have been used:

Breathing rate, spinal cord m y elopathy, bl adder function, tear production, etc.

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Note the dramatic sparing from fractionation

Lethality (LD 50 ass a y)

 LD 50 (mean lethal dose) = dose required to kill 50% of the animals in a given tim e period, e.g., 30 days, LD 50/30

 Usually im plies total body irradiation.

 Can also be deter m ined after irra diation of a portion of the body.

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Mortality of rhesus m onkeys following single total-body exposure t o x rays.

Death is from bone marrow depletion.

Multifraction Experiments

Early responding tissues : skin, intestinal epithelium, bone marrow These tissues are rapidly divi ding, self-renewing system s.

Late responding tissues: spinal cord, lung, kidney

“Radiation response of all tissues results from depletion of critical parenchymal cells.” Hall, 2000

Early/late differences are a functi on of the critical cell turnove r rate. Unanswered question: role of vascular damage vs parenchymal ce ll d e pletion.

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This approach allows estim a tio n of  /  ratios in tissues with non-clonogenic endpoint s.